International Journal of Molecular Sciences Article Yes-Associated Protein 1 Is a Novel Calcium Sensing Receptor Target in Human Parathyroid Tumors Giulia Stefania Tavanti 1,2,† , Chiara Verdelli 1,†, Annamaria Morotti 3,† , Paola Maroni 4 , Vito Guarnieri 5, Alfredo Scillitani 6, Rosamaria Silipigni 7, Silvana Guerneri 7, Riccardo Maggiore 8 , Gilberto Mari 8, Leonardo Vicentini 9, Paolo Dalino Ciaramella 10, Valentina Vaira 3,11 and Sabrina Corbetta 2,12,* 1 Laboratory of Experimental Endocrinology, IRCCS Istituto Ortopedico Galeazzi, 20161 Milan, Italy; [email protected] (G.S.T.); [email protected] (C.V.) 2 Department of Biomedical, Surgical and Dental Sciences, University of Milan, 20122 Milan, Italy 3 Division of Pathology, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, 20122 Milan, Italy; [email protected] (A.M.); [email protected] (V.V.) 4 Laboratory of Experimental Biochemistry & Molecular Biology, IRCCS Istituto Ortopedico Galeazzi, 20161 Milan, Italy; [email protected] 5 Division of Medical Genetics, Fondazione IRCCS Ospedale Casa Sollievo della Sofferenza, 71013 San Giovanni Rotondo, Italy; [email protected] 6 Endocrinology Unit, IRCCS Ospedale Casa Sollievo della Sofferenza, 71013 San Giovanni Rotondo, Italy; [email protected] 7 Medical Genetics Laboratory, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, 20122 Milan, Italy; [email protected] (R.S.); [email protected] (S.G.) 8 Endocrine Surgery, IRCCS Ospedale San Raffaele, 20123 Milan, Italy; [email protected] (R.M.); [email protected] (G.M.) 9 Endocrine Surgery, IRCCS Istituto Auxologico, 20122 Milan, Italy; [email protected] 10 Endocrinology Unit, ASST Grande Ospedale Metropolitano Niguarda, 20162 Milan, Italy; [email protected] Citation: Tavanti, G.S.; Verdelli, C.; 11 Department of Pathophysiology and Organ Transplantation, University of Milan, 20122 Milan, Italy Morotti, A.; Maroni, P.; Guarnieri, V.; 12 Endocrinology and Diabetology Service, IRCCS Istituto Ortopedico Galeazzi, 20161 Milan, Italy Scillitani, A.; Silipigni, R.; Guerneri, * Correspondence: [email protected]; Tel.: +39-02-6621-4647 S.; Maggiore, R.; Mari, G.; et al. † Those authors contribute equally to this work. Yes-Associated Protein 1 Is a Novel Abstract: The Hippo pathway is involved in human tumorigenesis and tissue repair. Here, we inves- Calcium Sensing Receptor Target in tigated the Hippo coactivator Yes-associated protein 1 (YAP1) and the kinase large tumor suppressor Human Parathyroid Tumors. Int. J. Mol. Sci. 2021, 22, 2016. https:// 1/2 (LATS1/2) in tumors of the parathyroid glands, which are almost invariably associated with doi.org/10.3390/ijms22042016 primary hyperparathyroidism. Compared with normal parathyroid glands, parathyroid adenomas (PAds) and carcinomas show variably but reduced nuclear YAP1 expression. The kinase LATS1/2, Academic Editor: Sang Yeol Lee which phosphorylates YAP1 thus promoting its degradation, was also variably reduced in PAds. Received: 24 December 2020 Further, YAP1 silencing reduces the expression of the key parathyroid oncosuppressor multiple Accepted: 16 February 2021 endocrine neoplasia type 1 (MEN1), while MEN1 silencing increases YAP1 expression. Treatment of Published: 18 February 2021 patient-derived PAds-primary cell cultures and Human embryonic kidney 293A (HEK293A) cells expressing the calcium-sensing receptor (CASR) with the CASR agonist R568 induces YAP1 nuclear Publisher’s Note: MDPI stays neutral accumulation. This effect was prevented by the incubation of the cells with RhoA/Rho-associated with regard to jurisdictional claims in coiled-coil-containing protein kinase (ROCK) inhibitors Y27632 and H1152. Lastly, CASR activation published maps and institutional affil- increased the expression of the YAP1 gene targets CYR61, CTGF, and WNT5A, and this effect was iations. blunted by YAP1 silencing. Concluding, here we provide preliminary evidence of the involvement of the Hippo pathway in human tumor parathyroid cells and of the existence of a CASR-ROCK-YAP1 axis. We propose a tumor suppressor role for YAP1 and LATS1/2 in parathyroid tumors. Copyright: © 2021 by the authors. Keywords: YAP1; LATS1/2; CASR; parathormone; MEN1; parathyroid tumors Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons 1. Introduction Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ The Hippo pathway is a central cellular pathway, which regulates homeostasis and 4.0/). plays prominent roles in carcinogenesis and regenerative processes. YAP1 (Yes-associated Int. J. Mol. Sci. 2021, 22, 2016. https://doi.org/10.3390/ijms22042016 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 2016 2 of 19 protein 1) and its paralog transcriptional coactivator with PDZ-binding motif, Tafazzin (TAZ), which are the principal effectors of the Hippo signaling pathway, are relatively inactive in adults to maintain organ homeostasis, while they are robustly induced during neoplastic disorders [1–3]. The Hippo-YAP/TAZ pathway has emerged as a hub that integrates diverse stimuli, including mechanical and cytoskeletal cues, cell adhesion, apico- basolateral polarity, and mitogens, to control cell growth and organ size [4]. The Hippo tumor suppressor pathway functions to inhibit the activity of YAP/TAZ transcriptional coactivators. The Hippo pathway is complex and involves the MST1 and MST2 (Mam- malian STE20-like protein kinase 1/2), LATS1 and LATS2 (large tumor suppressors 1 and 2), and adaptor proteins SAV1 (Salvador homolog 1) and MOB kinase activator 1A and 1B (MOB1A and MOB1B). MST1 and MST2 facilitate LATS1 and LATS2 phosphorylation, which in turn facilitate LATS-dependent phosphorylation of YAP and TAZ [5]. Genetic and epigenetic alterations of the tumor suppressor LATS1/2 have been observed in several cancers [6]. Phosphorylation of the complex YAP/TAZ results in degradation through ubiquitination or cytoplasmic retention via 14-3-3 binding; moreover, it further promotes β- TrCP-mediated YAP/TAZ ubiquitination and degradation. Therefore, upon the inhibition of the Hippo pathway, YAP/TAZ are activated and translocate into the nucleus to bind the TEAD (transcriptional enhanced associate domain) family of transcription factors, thus promoting cell proliferation, stem cell self-renewal, and tumorigenesis [7]. Parathyroid tumors, which are mainly benign, are the second most frequent endocrine neoplasia clinically associated with the endocrine disorder known as primary hyperparathy- roidism, which represents a frequently occurring cause of osteoporosis and bone fragility. Primary hyperparathyroidism is characterized by hypercalcemia and concomitant inappro- priately elevated parathormone (PTH) release from tumors of the parathyroid glands. The calcium-sensing receptor (CASR) is a key molecule involved in the exquisite 2+ parathyroid function of sensing extracellular calcium concentrations ([Ca ]o) and con- sequently of modulating PTH release and synthesis. CASR is a pleiotropic, type III G protein-coupled receptor (GPCR) that functionally associates with the cytoskeletal pro- tein filamin A [8,9]. The CASR has been shown to activate a wide range of intracellular pathways through coupling to different Gα proteins in a tissue- and cell-type-specific manner [8]. CASR is mainly coupled to Gα protein q/11 in parathyroid cells [10], through which the extracellular signal-regulated kinase (ERK) signaling activates [8,11]. CASR has also been reported to activate the small GTPase protein RhoA, presumably through Gα 12/13 and/or Gαq/11 [12–14]. The Hippo pathway has never been investigated in parathyroid tumors until now, while some evidence suggests its potential role in parathyroid tumorigenesis: (1) YAP1 gene maps on chromosome 11 in 11q22.1, a region frequently affected by the loss of het- erozygosity in parathyroid tumors [15,16]; (2) recent experimental data identified LATS2 gene as a target of the aberrantly expressed miR-372, which is overexpressed in a subset of parathyroid tumors [17]; (3) CASR, a crucial molecule in parathyroid tumors, might be coupled to Hippo signaling through RhoA/Rho-associated protein kinase (ROCK) activa- tion [12]. Recent studies uncovered the critical role of G protein-coupled receptors (GPCR) signaling in YAP/TAZ regulation [18–21]. ROCK is identified as a critical downstream effector of GTPase RhoA, which contains two isoforms, ROCK1 and ROCK2. ROCKs have a principal function in the generation of actin–myosin contractility and regulation of actin cytoskeleton dynamics. Moreover, they regulate various cellular functions, such as apoptosis, growth, migration, metabolism, and cellular contraction [22]. Here, we analyzed the expression and function of the Hippo pathway master regulator YAP1 in human parathyroid tissues and its interconnection with the parathyroid key genes multiple endocrine neoplasia type 1 (MEN1) and CASR. Int. J. Mol. Sci. 2021, 22, 2016 3 of 19 2. Results 2.1. YA1P Expression in Human Parathyroid Tumors The expression of the Hippo cofactor YAP1 was investigated by immunohistochem- istry in formalin-fixed paraffin-embedded (FFPE) sections from normal parathyroid glands (PaNs, n = 4), parathyroid adenomas (PAds, n = 11), and parathyroid carcinomas (PCas, n = 6) (Figure1). Normal parathyroid glands from normocalcemic patients showed that a consistent subset of the parathyroid